Compositions and methods for detecting, treating, and protecting against Fusobacterium infection

ABSTRACT

The present invention relates to protecting against, treating, and detecting  Fusobacteria  infections. Compositions and methods derived from nucleic acid and protein sequences of a 40 kDa Adhesin protein are provided to protect against, treat, and detect  Fusobacteria  infections in a subject. In one aspect, vaccines capable of inducing an immune response to a 40 kDa Adhesin protein are used to protect against  Fusobacteria  infection. Also, nucleic acid molecules, proteins, immunogens, antibodies, and antisense molecules derived from the sequences of the 40 kDa Adhesin protein may be used to protect against, treat, and detect  Fusobacteria  infections in a subject.

FIELD OF THE INVENTION

The present invention relates generally to the field of vaccines forgenerating protection from infectious disease and infection, andtreating symptoms and infections caused by infectious agents, as well asdetecting infection of a subject. More specifically, the presentinvention is useful for protection and treatment of infection byFusobacterium species.

BACKGROUND OF THE INVENTION

Fusobacterium necrophorum, a gram negative and rod-shaped anaerobe, isthe primary etiologic agent of liver abscesses in ruminant animals,including cattle and sheep. F. necrophorum strains are divided into twosubspecies, subsp. necrophorum (FNN) and subsp. funduliforme (FNF). Inaddition to liver abscesses, the organism is also the primary etiologicagent of foot rot, foot abscesses, calf diphtheria, and is frequentlyisolated from cases of mastitis, metritis, and necrotic lesions of theoral cavity. It has also been recognized as a human pathogen since thelate 1800s. Antibiotics have been used in animal agriculture to treatand prevent fusobacterial infections.

The increasing concern over the use of antibiotics in food animals andits impact on rise of multidrug resistant bacteria has already led tothe ban of certain antibiotics in animals. The sub-therapeutic use(i.e., use in the absence of disease) for growth promotion is a practicethat is becoming increasingly controversial, since it is implicated asincreasing antibiotic resistance among pathogenic bacteria of animalsand humans. Additionally, there is a growing demand for natural andorganic beef (requirements include cattle be raised without use ofantibiotics in feed), which warrants effective vaccines as a method toprevent infections. Accordingly, there is a need to develop an effectivevaccine for the prevention of fusobacterial infections, particularlyliver abscesses and foot rot.

U.S. Pat. Nos. 5,455,034, 5,492,694, and 5,861,162 describe leukotoxoidvaccines that immunize cattle and sheep against leukotoxin, an importantvirulence factor, released by F. necrophorum after it gains access intothe ruminal wall and the liver. While this vaccine reduces the severityand incidence of the disease (by up to 40%) it does not eliminate itsoccurrence. Moreover, leukotoxoid and bacterin vaccines frequentlycontain lipopolysaccharides and other cellular derivatives that haveside effects (fever, injection site abscesses, etc) and may impactfeed-intake and weight gain.

Accordingly, a need still exists for effective prevention and treatmentof fusobacterial infections. The present invention provides compositionsand methods for effective prevention and treatment of fusobacterialinfections.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods foreffectively preventing and treating FusobacteriumI infections in asubject. The present invention also relates detecting Fusobacteriuminfections.

In one embodiment, the compositions of the invention include isolatednucleic acid molecules. Suitable nucleic acid molecules have a sequencethat is at least 60% homologous to SEQ ID NO:1. In some embodiments, thenucleic acid molecule encodes the 40 kDa Adhesin protein (“Adhesin”) ofSEQ ID NO: 2. In some embodiments, the nucleic acid molecule encodes aportion of the Adhesin protein. Such partial coding may be used asantisense sequence to inhibit Adhesin protein, as molecular probes todetect Adhesin protein, or as primers in cloning techniques. Preferably,the nucleic acid molecule has a sequence that is about 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99%, or more homologous to the sequence of SEQ ID NO: 1.More preferably, the nucleic acid molecule has a sequence that is atleast 65% homologous to SEQ ID NO: 1.

The nucleic acid molecules of the invention may be within a vector.Suitable vectors include those used for cloning, expression, andpropagating.

In some embodiments the nucleic acid molecules encode a protein, orportion thereof, having at least 60% sequence homology with SEQ ID NO:2,or portion thereof. Preferably, the nucleic acid molecules encode aprotein, or portion thereof, that is about 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99%, or more homologous to the sequence of SEQ ID NO: 2, or portionthereof. More preferably, the nucleic acid molecules encode a protein,or portion thereof, that is at least 65% homologous to SEQ ID NO: 2, orportion thereof.

In another embodiment, the compositions of the invention include animmunogenic composition containing an isolated protein having at least60% sequence homology to SEQ ID NO:2 and a pharmaceutically acceptablecarrier. The isolated protein may encode a partial protein sequence.Such a partial sequence may be 5 to 356 amino acids in length.Preferably, the partial protein sequence is capable of inducing animmune response in a subject. Such partial peptide sequences of SEQ IDNO:2 include any partial sequence of SEQ ID NO:2 as well as those of SEQID NO: 3-5 and 8-17. Preferably, the partial sequence is SEQ ID NO: 9.

In some embodiments, the immunogenic composition includes a leukotoxinimmunogen. Suitable leukotoxin immunogens include recombinant leukotoxinprotein, partial recombinant leukotoxin peptides, leukotoxin proteinisolated from secretions of Fusobacterium and combinations thereof.

In one aspect, the present invention includes methods of immunizing asubject against Fusobacterium infection. In one embodiment, the methodsof immunizing include a method of inducing an immune response in asubject specific for infection by Fusobacterium where a subject isadministered an Adhesin agent. Suitable Adhesin agents includeantibodies, immunogens, antisense molecules, small molecules, orcombinations thereof. Suitable immunogens include isolated proteinshaving at least 60% sequence homology with SEQ ID NO: 2 or partialpeptide sequences thereof. Such partial peptide sequences of SEQ ID NO:2include any partial sequence of SEQ ID NO:2 as well as those of SEQ IDNO: 3-5 and 8-17. In another embodiment, the invention provides a methodof reducing the incidence of or severity of clinical signs or symptomscaused by infection by Fusobacterium where a subject is administered anAdhesin agent. The Adhesin agent may be provided in a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutically acceptablecarrier includes an adjuvant. In some embodiments, the subject is notinfected with Fusobacterium. In other embodiments, the subject isinfected with Fusobacterium. In some embodiments, the incidence ofsymptoms is reduced an amount ranging from about 5% to 100% whencompared to a control not receiving an Adhesin agent. Preferably, theincidence of symptoms is reduced an amount of about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more whencompared to a control not receiving an Adhesin agent. More preferably,the incidence of symptoms is reduced an amount of about 10% whencompared to a control not receiving an Adhesin agent. In someembodiments, the incidence of symptoms is reduced as to a specificsubject. In some embodiments, the incidence of symptoms is reduced as toa group of subjects. In some embodiments, the incidence of symptoms isreduced as to a herd of subjects.

In another aspect, the present invention includes methods of decreasingthe mortality rate of infection with Fusobacterium where a subject isadministered an Adhesin agent. The Adhesin agent may be provided in apharmaceutically acceptable carrier. In some embodiments, thepharmaceutically acceptable carrier includes an adjuvant. In someembodiments, the subject is not infected with Fusobacterium. In otherembodiments, the subject is infected with Fusobacterium. In someembodiments, the mortality rate is decreased about 5% to 100% whencompared to a control not receiving an Adhesin agent. Preferably, themortality rate is decreased about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95% or more when compared to a controlnot receiving an Adhesin agent. More preferably, the mortality rate isdecreased about 10% when compared to a control not receiving an Adhesinagent. In some embodiments, the mortality rate is decreased as to aspecific subject. In some embodiments, the mortality rate is decreasedas to a group of subjects. In some embodiments, the mortality rate isdecreased is reduced as to a herd of subjects.

In another aspect, the present invention includes methods of detectinginfection with Fusobacterium. Methods of detecting Fusobacteriuminfection include obtaining a sample from a subject, contacting thesample with an Adhesin indicator, and detecting the Adhesin indicator.Suitable Adhesin indicators include proteins, antibodies, antisensemolecules, and small molecules capable of binding to Adhesin or a codingsequence thereof. Preferably, the Adhesin indicators are labeled with adetection label, such as a fluorescence label, radiotype label, chemicallabel, or other label known or used in the art with detection assays.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 graphically illustrates the binding between subsp. necrophorum(FNN) (1) and subsp. funduliforme (FNF) (2) to bovine adrenal capillaryendothelial (EJG) cells;

FIG. 2A depicts transmission electron microscope (TEM) images(negatively stained) of Fusobacterium necrophorum subsp. necrophorum(FNN) from cattle;

FIG. 2B depicts transmission electron microscope (TEM) images(positively stained) of Fusobacterium necrophorum subsp. necrophorum(FNN) from cattle;

FIG. 2C depicts transmission electron microscope (TEM) images(negatively stained) of Fusobacterium necrophorum subsp. funduliforme(FNF) isolates from humans;

FIG. 2D depicts transmission electron microscope (TEM) images(positively stained) of Fusobacterium necrophorum subsp. funduliforme(FNF) isolates from humans;

FIG. 3 shows the outer membrane protein (OMP) profiles of subsp.Necrophorum (FNN) (Lanes 1 and 2) and subspecies funduliforme (FNF)(Lanes 4 and 5; Lane 3 is a molecular weight marker);

FIG. 4 graphically illustrates the attachment of FNN to EJG cells (1).The attachment reduced significantly when pretreated with polyclonalantisera raised against the OMPs of FNN (2). The attachment did notreduce when pretreated with polyclonal antisera raised against OMPs ofFNF (3). The attachment reduced significantly when EJG cells werepretreated with subsp. necrophorum OMPs (4) or with 40 kDa adhesin (5);

FIG. 5 shows an SDS-PAGE of serial steps in purification of a 40 kDaadhesin from cattle strain of FNN. Key: Lane 2, total unbound OMPremoved from the well; Lane 3, PBS wash; Lane 4, PBS+NP40; Lane 5,Modified RIPA; Lane 6, SDS sample buffer; Lane 8, western-blot of lane 6with antisera for OMPs of cattle FNN; Lane 9, western-blot with antiserafor OMPs of cattle FNF; Lane 11, far-western with biotinylatedfibronectin; and, Lanes 1, 7 and 10 are molecular weight markers;

FIG. 6 shows the 40 kDa adhesion is present in human F. necrophorumstrains. Key: Lane 1, 1 kb DNA ladder; Lanes 2-5, human strains; Lane 6,subsp. necrophorum, cattle; Lane 7, subsp. funduliforme, cattle; andLane 8, negative control;

FIG. 7 shows a western blot confirming the expression of 40 kDa adhesinin E. coli BL21(DE3) cells transfected with a vector containing the 40kDa coding sequence (SEQ ID NO: 6). Key: Lane 1, uninduced E. coli;Lanes 2-6, induced E. coli expressing the encoded 40 kDa protein;

FIG. 8 shows the 40 kDa adhesion protein adheres to EJG cells. The EJGcells are the polygonal cells containing dark round nuclei. The 40 kDais the bright white blurred spots covering the polygonal cells;

FIG. 9 shows attachment of F. necrophorum subsp. necrophorum (FNN) toBovine Capillary Endothelial (EJG) cells. The EJG cells are polygonalshaped cells with dark round nuclei contained within. The attached FNNare dark spots on the EJG cells;

FIG. 10 shows an SDS-PAGE of serial steps in purification of a 40 kDaadhesion. Key: Lane 2, Total unbound OMP removed from the well; PBSwash, Lane 3 treated, Lane 4 untreated; PBS+NP40, Lane 5 treated, Lane 6untreated; Modified RIPA, Lane 7 treated, Lane 8 untreated; SDS samplebuffer, Lane 10 treated, Lane 11 untreated; and, Lane 1 marker;

FIG. 11A shows the presence of 40 kDa adhesin in different strains ofsubsp. necrophorum (Lane 1:A25, Lane 2: A21, Lane 3: A50, Lane 4: 8L1)and subsp. funduliforme (Lane 6: B17, Lane 7:B29, Lane 8:B47, Lane 9:B35). Lane 5 is a 1 kb DNA ladder;

FIG. 11B shows the presence of 40 kDa adhesin in human F. necrophorumstrains (Lane 1: 1 kb DNA ladder, Lanes 2-5: human strains, Lane 6:subsp. necrophorum, Lane 7: subsp. funduliforme, and Lane 8: negativecontrol);

FIG. 12 shows the presence of fibronectin in EJG cell culture. The darkovals represent DAPI staining of cell nuclei, while the white elongatedstaining is the fibronectin;

FIG. 13 shows a Far-western analysis of the 40 kDa FncA Adhesin withbovine fibronectin. The biotinylated fibronectin bound to the purifiedFncA (1) and not the control (2);

FIG. 14 shows an SDS-PAGE of serial steps in purification of a 40 kDaadhesion. Key: Lane 2, Total OMP removed from the well; 3, PBS wash; 4,PBS+NP40; 5, Modified RIPA; 6, SDS sample buffer; 8, western blot oflane 6 with antisera for total OMPs of FNN; 9, western blot of lane 6with antisera for total OMPs of FNF; Lanes 1 and 7 are molecular weightmarkers;

FIG. 15 graphically illustrates that attachment of FNN to EJG cells wasreduced significantly when FNN was pretreated with antiserum raisedagainst the 40 kDa adhesion of FNN (3) compared to no serum (1) orpretreatment with pre-vaccination serum (2);

FIG. 16 shows a 2D representation of 40 kDa adhesin using Viterbimethod;

FIG. 17 shows a 2D representation of 40 kDa adhesin using N-best method;

FIG. 18 shows a 2D representation of 40 kDa adhesin using a posteriordecoding method;

FIG. 19 shows large abscesses (white areas) in the liver of a mouse(from no-vaccine control) infected intra-peritoneally with F.necrophorum; and,

FIG. 20 shows the isolation of F. necrophorum as pure cultures from theliver, lung, and spleen from a no-vaccine control mouse.

DETAILED DESCRIPTION

Applicants have discovered a protein that plays a pivotal role inmediating Fusobacterium attachment to host cells allowing pathologicinfection of a host. The present invention encompasses this discoveryand provides compositions and methods based on the discovered proteinand attachment mechanism. In particular, the present invention providescompositions and methods useful in research, diagnostics, andtherapeutics for conditions and diseases associated with pathologicinfection of Fusobacterium necrophorum. The compositions and methods aredirected at inducing an immune response in a subject to protect against,or treat, Fusobacterium infection, as well as detection ofFusobacterium.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Compositions

A. Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode Adhesin proteins or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify Adhesin-encoding nucleic acids (e.g., Adhesin mRNA)and fragments for use as PCR primers for the amplification or mutationof Adhesin nucleic acid molecules.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or a complementthereof, may be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or portion of thenucleic acid sequence of SEQ ID NO: 1, Adhesin nucleic acid moleculesmay be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook et al., eds., Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid of the invention may be amplified using cDNA, mRNA orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified may be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toAdhesin nucleotide sequences may be prepared by standard synthetictechniques known in the art, such as using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, or portion thereof. A nucleicacid molecule which is complementary to a given nucleotide sequence isone which is sufficiently complementary to the given nucleotide sequencethat it can hybridize to the given nucleotide sequence thereby forming astable duplex.

Moreover, the nucleic acid molecule of the invention may comprise only aportion of a nucleic acid sequence encoding Adhesin. By way of example,a fragment of the nucleic acid coding sequence can be used as a probe,primer, or a fragment encoding a biologically active portion of Adhesin.The nucleotide sequence determined from the cloning of the Adhesin geneallows for the generation of probes and primers designed for use inidentifying and/or cloning Adhesin homologues in other cell types, aswell as Adhesin homologues and orthologs from other mammals. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; preferably about 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, or 49; more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense orantisense sequence of SEQ ID NO:1, or of a naturally occurring mutant ofSEQ ID NO:1.

Probes based on the Adhesin nucleotide sequence may be used to detecttranscripts or genomic sequences encoding the same or similar proteins.The probe comprises a label group attached thereto, such as aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes may be used in diagnostic or screening assays and includethose known in the art or yet to be discovered.

A nucleic acid fragment encoding a “biologically active portion” ofAdhesin may be prepared by isolating a portion of SEQ ID NO:1, whichencodes a polypeptide having a Adhesin biological activity, expressingthe encoded portion of Adhesin protein (e.g., by recombinant expressionin vitro) and assessing the activity of the encoded portion of Adhesin.For example, a nucleic acid fragment encoding a biologically activeportion of Adhesin includes a host cell binding domain. Suitable nucleicacid fragments may encode biologically active portions of Adhesin suchas those of SEQ ID NO: 3-5 and 8-17.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO:1, due to degeneracy of thegenetic code and thus encode the same Adhesin protein as that encoded bythe nucleotide sequence shown in SEQ ID NO:1.

In addition to the Adhesin nucleotide sequence shown in SEQ ID NO:1, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences ofAdhesin may exist. Such genetic polymorphism in the Adhesin gene mayexist among bacteria within a subspecies due to natural allelicvariation. Such natural allelic variations typically result in 15%variance in the nucleotide sequence of the Adhesin gene. Any and allsuch nucleotide variations and resulting amino acid polymorphisms inAdhesin that are the result of natural allelic variation and that do notalter the functional activity of Adhesin are intended to be within thescope of the invention. Thus, e.g., 1%, 2%, 3%, 4%, or 5% of the aminoacids in Adhesin (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40,45, 50, or 55 amino acids) may be replaced by another amino acid,preferably by conservative substitution.

Moreover, nucleic acid molecules encoding Adhesin proteins from otherspecies (Adhesin orthologs/homologues), which have a nucleotide sequencewhich differs from that of an Adhesin disclosed herein, are intended tobe within the scope of the invention.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least about 12 to 1075 (15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 325, 350,375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, and 1071)nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence, preferably thecoding sequence, of SEQ ID NO:1.

In addition to naturally occurring allelic variants of the Adhesinsequence that may exist in the population, the skilled artisan willfurther appreciate that changes may be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, thereby leading to changes in theamino acid sequence of the encoded protein without altering thefunctional ability of the protein. For example, such mutations mayinclude nucleotide substitutions leading to amino acid substitutions at“unnecessary” amino acid residues. An “unnecessary” amino acid residueis a residue that may be altered from the wildtype sequence of Adhesinprotein without altering the biological activity, whereas an “necessary”amino acid residue is required for biological activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding Adhesin proteins that contain changes in amino acidresidues that may or may not be essential for activity. Such Adhesinproteins differ in amino acid sequence from SEQ ID NO: 2. In oneembodiment, the isolated nucleic acid molecule includes a nucleotidesequence encoding a protein that includes an amino acid sequence that isat least about 45% identical, 65%, 75%, 85%, 95%, 98% or more identicalto the amino acid sequence of SEQ ID NO:2. An isolated nucleic acidmolecule encoding an Adhesin protein having a sequence which differsfrom that of SEQ ID NO:2, may be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of Adhesin (SEQ ID NO:1) such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations may be introduced by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.

The present invention encompasses antisense nucleic acid molecules.Antisense molecules are complementary to a sense nucleic acid encoding aprotein, complementary to the coding strand of a double-stranded cDNAmolecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid hydrogen bonds to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire Adhesin codingstrand, or to only a portion thereof, such as all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding Adhesin. The noncoding regions (“5′ and3′ untranslated regions”) are the 5′ and 3′ sequences that flank thecoding region and are not translated into amino acids. Given the codingstrand sequences encoding Adhesin disclosed herein, antisense nucleicacids of the invention may be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule may becomplementary to the entire coding region of Adhesin mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of Adhesin mRNA. For example, theantisense oligonucleotide may be complementary to the region surroundingthe translation start site of Adhesin mRNA. An antisense oligonucleotidemay be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention may beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) may be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which may be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-aino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid may beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an Adhesinprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization may be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An antisense nucleic acid molecule of the inventionmay be administered by direct injection at a tissue site. Alternatively,antisense nucleic acid molecules may be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules may be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules may also be delivered to cellsusing the plasmids described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, plasmid constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

B. Adhesin Proteins

One aspect of the invention pertains to isolated Adhesin proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-Adhesin antibodies. In oneembodiment, native Adhesin proteins may be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, Adhesin proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, an Adhesin protein or polypeptide may be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theAdhesin protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofAdhesin protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, Adhesin protein that is substantially free of cellularmaterial includes preparations of Adhesin protein having less than about30%, 20%, 10%, or 5% (by dry weight) of non-Adhesin protein (alsoreferred to herein as a “contaminating protein”). When the Adhesinprotein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When Adhesin protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly, such preparations of Adhesin protein have lessthan about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors ornon-Adhesin chemicals.

Biologically active portions of a Adhesin protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the Adhesin protein (e.g., the aminoacid sequence shown in SEQ ID NO:2), which include less amino acids thanthe full length Adhesin protein, and exhibit at least one activity of aAdhesin protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the Adhesin protein. Abiologically active portion of an Adhesin protein may be a polypeptidewhich is, for example, 10, 25, 50, 100, 150, 200, 250, 300 or more aminoacids in length. Preferred biologically active polypeptides include oneor more identified Adhesin structural domains, such as host cellattachment domain and other domains that may be discovered. Suchbiologically active portions of Adhesin protein are those of SEQ ID NO:3-5 and 8-17.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, may be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeAdhesin protein.

The Adhesin protein has the amino acid sequence of SEQ ID NO:2. Otheruseful Adhesin proteins are substantially identical to SEQ ID NO:2 andretain the functional activity of the protein of SEQ ID NO:2, yet differin amino acid sequence due to natural allelic variation or mutagenesis.

A useful Adhesin protein is a protein which includes an amino acidsequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%identical to the amino acid sequences of SEQ ID NO:2, 3-5, or 8-17. Insome embodiments, a useful Adhesin protein is a protein which includesan amino acid sequence at least about 45%, preferably 55%, 65%, 75%,85%, 95%, or 99% identical to the amino acid sequences of SEQ ID NO:2,3-5, or 8-17 and retains the functional activity of the Adhesin proteinof SEQ ID NO:2, 3-5, or 8-17. Preferably, the sequence is about 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to theamino acid sequence of SEQ ID NO:2, 3-5, or 8-17. More preferably, thesequence is about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70% or more identical to the amino acid sequence of SEQ ID NO:2,3-5, or 8-17. More preferably, the sequence is about 60% identical tothe amino acid sequence of SEQ ID NO:2, 3-5, or 8-17.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions.times.100).

The determination of percent homology between two sequences may beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'lAcad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences similar or homologous to Adhesin nucleic acidmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

The invention also provides Adhesin chimeric or fusion proteins. As usedherein, an Adhesin “chimeric protein” or “fusion protein” comprises anAdhesin polypeptide operatively linked to a non-Adhesin polypeptide. An“Adhesin polypeptide” refers to a polypeptide having an amino acidsequence corresponding to all or a portion (preferably a biologicallyactive portion) of a Adhesin, whereas a “non-Adhesin polypeptide” refersto a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially identical to a Adhesin protein.Within the fusion protein, the term “operatively linked” is intended toindicate that the Adhesin polypeptide and the non-Adhesin polypeptideare fused in-frame to each other. The heterologous polypeptide may befused to the N-terminus or C-terminus of the Adhesin polypeptide.

One useful fusion protein is a GST fusion protein in which the Adhesinsequences are fused to the C-terminus of the GST sequences. Such fusionproteins can facilitate the purification of recombinant Adhesin.

In yet another embodiment, the fusion protein is anAdhesin-immunoglobulin fusion protein in which all or part of Adhesin isfused to sequences derived from a member of the immunoglobulin proteinfamily. The Adhesin-immunoglobulin fusion proteins of the invention maybe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between Adhesin protein and Adhesintargets, such as a host cell, to thereby suppress Adhesin activity invivo. Inhibition of the Adhesin target/Adhesin interaction may be usefultherapeutically for both the treatment of bacterial infections ofFusobacterium, as well as prevention of pathologic disease caused byFusobacterium. Moreover, the Adhesin-immunoglobulin fusion proteins ofthe invention may be used as immunogens to produce anti-Adhesinantibodies in a subject, to purify Adhesin ligands, in detection assaysto detect the presence of Fusobacterium and in screening assays toidentify molecules which inhibit the interaction of Adhesin with anAdhesin target.

Preferably, an Adhesin chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques. Suitabletechniques include by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene may be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments may be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons:1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnAdhesin-encoding nucleic acid may be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the Adhesinprotein.

The present invention also pertains to variants of the Adhesin proteinswhich function as Adhesin antagonists. Variants of the Adhesin proteinsmay be generated by mutagenesis techniques known in the art. Anantagonist of the Adhesin protein may inhibit one or more of theactivities of the naturally occurring form of the Adhesin protein by,for example, competitively binding to a downstream or upstream member ofa cellular signaling cascade which includes the Adhesin protein, or byinhibiting binding to Adhesin targets. Thus, specific biological effectsmay be elicited by treatment with a variant of limited function.Treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the protein may have fewerside effects in a subject relative to treatment with the naturallyoccurring form of the Adhesin proteins.

Variants of the Adhesin protein which function as Adhesin antagonistscan be identified by screening combinatorial libraries of mutants, suchas truncation mutants of the Adhesin protein for Adhesin antagonistactivity. In one embodiment, a variegated library of Adhesin variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of Adhesinvariants may be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential Adhesin sequences is expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of Adhesinsequences therein. There are a variety of methods which may be used toproduce libraries of potential Adhesin variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence may be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential Adhesinsequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura etal. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the Adhesin protein codingsequence can be used to generate a variegated population of Adhesinfragments for screening and subsequent selection of variants of aAdhesin protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa Adhesin coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal and internal fragments of various sizes of theAdhesin protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of Adhesin proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify Adhesinvariants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

C. Expression Systems

In one aspect, the sequences of the invention may be incorporated intoan expression system. Many expression systems are known in the art andare contemplated herein. Suitable expression systems include those thatuse an expression vector. Suitable expression vectors include regulatorysequences, operably linked to the sequences of the invention, thatpermit transcription and translation of the coding sequence. Exemplaryregulatory sequences include, without limitation, promoters, enhancers,terminators, operators, repressors, inducers, and other regulatoryelements known in the art. Expression vectors generally have convenientrestriction sites located near the promoter sequence to provide for theinsertion of nucleic acid sequences. Also, selectable markers operativein the expression host may be included in the expression vector. Anyexpression vector known in the art or yet to be discovered may be usedwith the coding sequences of the invention. A skilled artisan willrecognize that the choice of vector for use with the invention isdependent on the intended use as well as the host within which thecoding sequence is to be expressed. Suitable vectors include, but arenot limited to, bacteriophage-derived vectors, viral vectors, retroviralvectors, adenoviral vectors, prokaryotic expression vectors, andeukaryotic expression vectors. In some embodiments, an expression vectorcapable of prokaryotic expression is preferred. In other embodiments, anexpression vector capable of eukaryotic expression is preferred. In yetother embodiments, an expression vector capable of both prokaryotic andeukaryotic expression is preferred. In one aspect, an inducibleexpression vector is preferred. Suitable inducible expression vectorsinclude those known in the art that activate transcription only in thepresence of a specific inducer (e.g. isopropyl thiogalactopyranoside(IPTG), hormone-based inducers such as progesterone, antibiotic-basedinducers such as tetracycline, etc.). Preferably, the expression vectorincludes a T7 promoter. Exemplary vectors include, without limitation,pET vectors, pET31b, pET32, pET22b, pLac1, pLysE, pLysS, and othersknown in the art. More preferably, the expression vector is pET22b.

Expression vectors may be used with any compatible host cell. Theexpression vector may exist in a host cell as an extrachromosomalelement or integrated into the host genome. Host cells may beprokaryotic, such as any number of bacteria strains, or may beeukaryotic, such as yeast or other fungal cells, insect, plant,amphibian, or mammalian cells, including rodent, animal or human cells.In one aspect, it is preferred that the host cells are prokaryotic.Suitable prokaryotic cells include a number of bacteria strainsincluding strains of E. coli. Preferably, the host cell is E. coli DH5α,E. coli BL21, or any other bacteria known in the art. More preferably,the host cell is E. coli BL21.

D. Antibodies

An isolated Adhesin protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind Adhesin usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length Adhesin protein can be used or, alternatively, theinvention provides antigenic peptide fragments of Adhesin for use asimmunogens. The antigenic peptide of Adhesin comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence shown in SEQ ID NO:2 and encompasses an epitope of Adhesin suchthat an antibody raised against the peptide forms a specific immunecomplex with Adhesin. Preferably, the antigenic peptide of Adhesincomprises at least 8 or more amino acids of sequences shown in SEQ IDNO: 2, 3-5, or 8-17. More preferably, the antigenic peptide of Adhesincomprises at least 8 or more amino acids of sequences shown in SEQ IDNO: 2.

Useful antibodies include antibodies which bind to a domain or subdomainof Adhesin described herein (e.g., attachment site).

An Adhesin immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation maycontain, for example, recombinantly expressed Adhesin protein or achemically synthesized Adhesin polypeptide. The preparation may furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic Adhesin preparation induces a polyclonalanti-Adhesin antibody response.

Accordingly, another aspect of the invention pertains to anti-Adhesinantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds an antigen, such as Adhesin. A molecule whichspecifically binds to Adhesin is a molecule which binds Adhesin, butdoes not substantially bind other molecules in a sample. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind Adhesin. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of Adhesin. A monoclonal antibody composition thustypically displays a single binding affinity for a particular Adhesinprotein with which it immunoreacts.

Polyclonal anti-Adhesin antibodies can be prepared as described above byimmunizing a suitable subject with an Adhesin immunogen. Theanti-Adhesin antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized Adhesin. If desired, theantibody molecules directed against Adhesin can be isolated from themammal (e.g., from the blood) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-Adhesinantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally Current Protocols in Immunology (1994) Coligan et al.(eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with an Adhesin immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds Adhesin.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-Adhesin monoclonal antibody (see, e.g., Current Protocols inImmunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth,in Monoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); and Lemer (1981) Yale J. Biol.Med., 54:387-402). Moreover, the ordinarily skilled artisan willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind Adhesin, e.g., using a standard ELISA assay.

Additionally, recombinant anti-Adhesin antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-Adhesin antibody (e.g., monoclonal antibody) can be used toisolate Adhesin by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-Adhesin antibody can facilitate thepurification of natural Adhesin from cells and of recombinantly producedAdhesin. Moreover, an anti-Adhesin antibody can be used to detectAdhesin protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the Adhesinprotein. Anti-Adhesin antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, forexample, to determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials known in the art.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes LUMINOL® detecting agent;examples of bioluminescent materials include luciferase, luciferin, andaequorin, and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response. The drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, a-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophase colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), or other growth factors. Furthermore,the conjugate may be any moiety useful in the use of the invention andinclude those known in the art or yet to be discovered. A skilledartisan will recognize that the conjugate used depends upon the intendeduse of the invention.

Techniques for conjugating such therapeutic moieties to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies for Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of CytotoxicAgents in Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological and Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, and Future Prospective of TheTherapeutic Use of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

In addition, antibodies of the invention, either conjugated or notconjugated to a therapeutic moiety, can be administered together or incombination with a therapeutic moiety such as a cytotoxin, a therapeuticagent or a radioactive metal ion. The order of administration of theantibody and therapeutic moiety can vary. For example, in someembodiments, the antibody is administered concurrently (through the sameor different delivery devices, e.g., syringes) with the therapeuticmoiety. Alternatively, the antibody can be administered separately andprior to the therapeutic moiety. Still alternatively, the therapeuticmoiety is administered separately and prior to the antibody. In manyembodiments, these administration regimens will be continued for days,months or years.

E. Pharmaceutical Compositions

The Adhesin nucleic acid molecules, Adhesin proteins, Adhesinimmunogens, small molecules, and anti-Adhesin antibodies (also referredto herein as “Adhesin agent”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein,antibody, immunogen, small molecules or combinations thereof and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and combinations thereof. Oneparticularly preferred composition includes saline, preferably phosphatebuffered saline. Another particularly preferred composition includesFreund's complete adjuvant. Yet another preferred composition includesboth phosphate buffered saline and Freund's complete adjuvant. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In some embodiments, the pharmaceutically acceptable carrier includesadjuvants. Suitable adjuvants are those that include surfactants, oil,mycobacterium, immunostimulators, zinc proline, detergent, modifiedbacterial products, other components known in the art, and anycombination thereof. Adjuvants commonly known in the art include,without limitation, Freund's Complete Adjuvant, Freund's IncompleteAdjuvant, Hunter's TITERMAXd® adjuvant, Gerbu Adjuvant, and Ribi'sAdjuvant. One skilled in the art will appreciate that the adjuvantchosen depends upon a variety of factors including, without limitation,the specific agent employed; the age, body weight, general health,gender, and species of the subject; the route of administration; anydrug combinations; and the degree of immune response desired.Preferably, the pharmaceutically acceptable carrier includes TITERMAX®adjuvant or Freund's Complete Adjuvant. More preferably, thepharmaceutically acceptable carrier includes TITERMAX® adjuvant.

The invention includes methods for preparing pharmaceutical compositionsfor treating or preventing an infection of Fusobacterium. Such methodscomprise formulating a pharmaceutically acceptable carrier with anAdhesin agent that is capable of inducing an immune response to Adhesinin the subject to be treated, inhibiting the activity of Adhesin, orcombinations thereof. Such compositions can further include additionalactive agents. Thus, the invention further includes methods forpreparing a pharmaceutical composition by formulating a pharmaceuticallyacceptable carrier with an Adhesin agent that is capable of inducing animmune response to Adhesin in the subject to be treated, an Adhesinagent capable of inhibiting the activity of Adhesin, additional activecompounds, and combinations thereof.

The Adhesin agent used in the pharmaceutical composition may be nucleicacids, protein, immunogens, or antibodies as described herein, or asmall molecule. Suitable small molecules include peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled artisan. The dose(s) of the small molecule will vary,for example, depending upon the identity, size, and condition of thesubject or sample being treated, further depending upon the route bywhich the composition is to be administered, if applicable, and theeffect which the practitioner desires the small molecule to have uponthe nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular subject willdepend upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, gender, anddiet of the subject, the time of administration, the route ofadministration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.Preferably, the parenteral preparation is enclosed in multiple dosevials.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOR®EL solubilizing agent (BASF; Parsippany, N.J.) or phosphate bufferedsaline (PBS). In all cases, the composition must be sterile and shouldbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193). Preferably, the dosage is about 0.5, 1, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65,70, 75, 80, 85, 90, or 95 mg/kg of body weight. More preferably, thedosage is about 10 to 20 mg/kg. More preferably, the dosage is about 15mg/kg.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.

The gene therapy vectors of the invention can be either viral ornon-viral. Examples of plasmid-based, non-viral vectors are discussed inHuang et al. (1999) Nonviral Vectors for Gene Therapy (supra). Amodified plasmid is one example of a non-viral gene delivery system.Peptides, proteins (including antibodies), and oligonucleotides may bestably conjugated to plasmid DNA by methods that do not interfere withthe transcriptional activity of the plasmid (Zelphati et al. (2000)BioTechniques 28:304-315). The attachment of proteins and/oroligonucleotides may influence the delivery and trafficking of theplasmid and thus render it a more effective pharmaceutical composition.

II. Methods

The present invention encompasses methods of detecting, treating, andpreventing fusobacterial infections in a subject. The methods may beutilized to treat a subject harboring symptoms that would benefit fromAdhesin compositions or that is at risk of developing a condition thatwould benefit from Adhesin compositions.

A. Conditions Benefiting from Adhesin Compositions

Conditions that would benefit from Adhesin compositions, such astreatment with inhibiting antibodies, Adhesin immunogens, Adhesinantisense molecules, small molecules, or proteins, may include anysymptom, condition or disease that is caused by fusobacterial infection.For instance, exemplary conditions that may benefit include liverabscess, foot rot, foot abscesses, calf diphtheria, mastitis, mertritis,necrotic lesions of the oral cavity, sore throat, pharyngitis,tonsillitis, deep neck abscesses, deep neck infections, metastaticpleuropulmonary disease, necrotizing pneumonia, empyema, renal andhepatic abscesses, osteomyelitis, meningitis paravertebral abscesses,and pustular dermatitis of hypoperfused extremities.

Also, methods of the invention may be utilized to treat a population ofcells that would benefit from Adhesin compositions. Such cells includethose in a subject as well as those removed from a subject fortherapeutic treatment, cultured cells, those used in gene-therapypractices, and any other cell that may benefit from Adhesincompositions.

B. Methods of the Invention

The Adhesin compositions described herein, including nucleic acidmolecules, proteins, antisense molecules, immunogens, and antibodies,may be used in detection assays and treatment methods (e.g., therapeuticand prophylactic) of the invention. The isolated nucleic acid moleculesof the invention can be used to express Adhesin protein, to detectAdhesin, and to modulate Adhesin activity. In addition, the Adhesinproteins can be used to screen drugs, compounds, or antibodies whichmodulate the Adhesin activity or expression as well as to treatfusobacterial infections. In addition, the anti-Adhesin antibodies ofthe invention can be used to detect and isolate Adhesin proteins as wellas modulate Adhesin activity. Also, Adhesin immunogens can beadministered to a subject to induce an immune response in a subject totreat or prevent fusobacterial infections, such as an Adhesin vaccine.Further, Adhesin antisense molecules can be used to modulate Adhesinactivity, such as inhibit Adhesin activity. This invention furtherpertains to novel agents identified by the above-described screeningassays and uses thereof for treatments as described herein.

1. Treatment Methods

In one embodiment, the Adhesin compositions described herein may be usedin methods of treating subjects infected with Fusobacteria. In anotherembodiment, the Adhesin compositions described herein may be used inmethods of treating subjects not infected with Fusobacteria. In anotherembodiment, the Adhesin compositions may be used to alleviate conditionscaused by, or related to Fusobacteria infection. Yet, in otherembodiments, the Adhesin compositions may be used to decrease mortalitycaused by Fusobacteria infection.

The Adhesin compositions may be administered to a subject in a singledose or multiple doses. A dosing regimen, either single or multipledoses, may be followed with a booster dose. The amount of time a boosterdose may follow a dosing regimen composition depends upon the efficacyof the dosing regimen.

In other embodiments, subjects being administered Adhesin compositionsof the invention may also be administered combination therapies, inwhich additional treatments are used. Such additional treatments includetherapeutic treatments known in the art, or yet to be discovered, thatprovide a benefit to the subject. For example, a subject undergoingAdhesin vaccination against fusobacterial infection may be administeredtherapeutics such as leukotoxin vaccine, immunomodulating agents, orantisense molecules. The additional therapeutics may be administeredindividually, sequentially, or in combination with other therapeutics orthe Adhesin composition.

In some embodiments, the efficacy of the Adhesin agents may be measuredby the effect on the agents on symptoms experienced by the recipientsubject. For example, a reduction in the incidence or severity, up toand including complete prevention, of symptoms of Fusobacterium can bemeasured to assess the efficacy of the Adhesin agents. Also, thepresence of Fusobacterium infection can be used to measure the efficacyof the Adhesin agents. Symptoms of Fusobacterium infection in humansinclude Lemiere's syndrome, postanginal sepsis, tonsillitis, septicthrombophlebitis of the internal jugular vein, septicemia, septicemboli, purulent otits, septic arthritis, cerebral abscess, cough, sorethroat, oropharyngeal sepsis, cervical lymphadenopathy, high level ofC-reactive protein, lung lesions, metastatic lesions, high fever,chills/rigor, and combinations thereof. Symptoms of Fusobacteriuminfection in animals include liver abscesses, foot rot, ruminalacidosis, rumenitis, thrush, reduced weight gain, reduced feedconversion efficiency, and combinations thereof. Methods of detectingand monitoring such symptoms are known in the art and contemplatedherein. In one embodiment, subjects prophylactically treated withAdhesin agents to prevent Fusobacterium infection can be monitored forthe presence of Fusobacterium infection and symptoms associatedtherewith. In one embodiment, subjects having symptoms of Fusobacteriuminfection can be monitored for alleviation of such symptoms. Preferably,there are no symptoms of Fusobacterium infection detected. Morepreferably, there is a reduction in incidence of symptoms by about 5-99%when compared to a control subject not receiving an Adhesin agent. Morepreferably, there is a reduction in incidence of symptoms by about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40% or more when compared toa control subject not receiving an Adhesin agent. More preferably, thereis a reduction in incidence of symptoms by about 10% when compared to acontrol subject not receiving an Adhesin agent. In one embodiment, thereduction in incidence of symptoms is detected as to a specific subject.In another embodiment, the reduction in incidence of symptoms isdetected as to a group of subjects. In another embodiment, the reductionin incidence of symptoms is detected as to a herd.

In some embodiments, the Adhesin agents can be administered to a subjectto decrease mortality caused by Fusobacterium infection. Preferably,mortality caused by Fusobacterium infection is eliminated. Morepreferably, there is a decrease in mortality rate of about 1-99% whencompared to a control subject not administered an Adhesin agent. Morepreferably, there is a decrease in mortality rate of about 5, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95% or more when compared to a control subject not receivingan Adhesin agent. More preferably, there is a decrease in mortality rateof about 10% when compared to a control subject not receiving an Adhesinagent. In one embodiment, the decrease in mortality rate is detected asto a specific subject. In another embodiment, the decrease in mortalityrate is detected as to a group of subjects. In another embodiment, thedecrease in mortality rate is detected as to a herd.

2. Detection Methods

Another aspect of the present invention relates to diagnostic assays fordetecting a fusobacterial infection, in the context of a biologicalsample (e.g., blood, serum, cells, tissue) to thereby determine whethera subject is infected with Fusobacterium. An exemplary method fordetecting the presence or absence of Adhesin in a sample involvesobtaining a sample from a test subject and contacting the sample with acompound or an agent capable of detecting Adhesin protein or nucleicacid (e.g., mRNA, genomic DNA) that encodes Adhesin protein such thatthe presence of Adhesin is detected in the sample. An agent fordetecting Adhesin mRNA or genomic DNA is a labeled nucleic acid probecapable of hybridizing to Adhesin mRNA or genomic DNA. The nucleic acidprobe can be, for example, a full-length Adhesin nucleic acid, such asthe nucleic acid of SEQ ID NO:1, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250, 500, 750 or morenucleotides in length and sufficient to specifically hybridize understringent conditions to mRNA or genomic DNA. Other suitable probes foruse in the diagnostic assays of the invention are described herein.

An agent for detecting Adhesin protein can be an antibody capable ofbinding to Adhesin protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The detection method of the invention can be used todetect Adhesin mRNA, protein, or genomic DNA in a sample in vitro aswell as in vivo. For example, in vitro techniques for detection ofAdhesin mRNA include Northern hybridizations and in situ hybridizations.In vitro techniques for detection of Adhesin protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of Adhesin genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of Adhesin protein includeintroducing into a subject a labeled anti-Adhesin antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the sample contains protein molecules from the testsubject. Alternatively, the biological sample can contain mRNA moleculesfrom the test subject or genomic DNA molecules from the test subject.

In another embodiment, the methods further involve obtaining a controlsample from a control subject, contacting the control sample with acompound or agent capable of detecting Adhesin protein, mRNA, or genomicDNA, such that the presence of Adhesin protein, mRNA or genomic DNA isdetected in the sample, and comparing the presence of Adhesin protein,mRNA or genomic DNA in the control sample with the presence of Adhesinprotein, mRNA or genomic DNA in the test sample.

C. Delivery Means and Routes

Methods of administration include any method known in the art or yet tobe discovered. Exemplary administration methods include intravenous,intraocular, intratracheal, intratumoral, oral, rectal, topical,intramuscular, intraarterial, intrahepatic, intrathoracic, intrathecal,intracranial, intraperitoneal, intrapancreatic, intrapulmonary, orsubcutaneously. Preferably, the method of administration isintramuscular.

Adhesin compositions of the invention are typically administered to asubject in an amount sufficient to provide a benefit to the targetmicroenvironment of the subject. This amount is defined as a“therapeutically effective amount.” The therapeutically effective amountwill be determined by the efficacy or potency of the particularcomposition, the duration or frequency of administration, and the sizeand condition of the subject, including that subject's particulartreatment response. Additionally, the route of administration should beconsidered when determining the therapeutically effective amount. It isanticipated that the therapeutically effective amount of an Adhesincomposition of the invention will range from about 0.1 ml/kg to about 35ml/kg. Preferably, the therapeutically effective amount of an Adhesincomposition of the invention ranges from 0.1 ml/kg to 1 ml/kg. Dependingon the target area and desired therapeutic agent used in conjunction (ofin certain instances no additional therapeutic agent will be used) withthe Adhesin composition the amount of Adhesin composition can include0.01%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the total therapeuticcomposition. In determining the therapeutically effective amounts, oneskilled in the art will also consider the existence, nature, and extentof any adverse effects that accompany the administration of a particularcompound in a particular subject.

III. Kits

The present invention provides articles of manufacture and kitscontaining materials useful for preventing, treating, or detectingfusobacterial infection. The article of manufacture may include acontainer of a composition as described herein with a label. Suitablecontainers include, for example, bottles, vials, and test tubes. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition having an active agent whichis effective for preventing, treating, or detecting fusobacterialinfection. The active agent is at least one Adhesin composition of theinvention and may further include additional Adhesin compositions orbioactive agents known in the art for treating the specific condition.

For antibody-based kits, the kit may comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to Adhesinprotein; and, optionally, (2) a second, different antibody which bindsto Adhesin protein or the first antibody and is conjugated to adetectable agent. For oligonucleotide-based kits, the kit may comprise,for example: (1) a oligonucleotide, (e.g., a detectably labeledoligonucleotide), which hybridizes to a Adhesin nucleic acid sequence or(2) a pair of primers useful for amplifying an Adhesin nucleic acidmolecule.

The kit may also comprise, a buffering agent, a preservative, or aprotein stabilizing agent. The kit may also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for use. The label on thecontainer may indicate that the composition is useful for preventing,treating, or detecting specific conditions and may also indicatedirections for administration.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications are incorporated by reference in their entirety. In theevent that there is a plurality of definitions for a term herein, thosein this section prevail unless stated otherwise.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A, non-limiting example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2.×SSC, 0.1% SDS at50-65° C. (e.g., 50° C. or 60° C. or 65° C.) Preferably, the isolatednucleic acid molecule of the invention that hybridizes under stringentconditions corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs in ahuman cell in nature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an Adhesinprotein, preferably 40 kDa Adhesin isolated from Fusobacteriumnecrophorum or variant thereof.

As used herein, the terms “protein” and “recombinant protein” refer toamino acid molecules comprising an Adhesin protein, preferably 40 kDaAdhesin isolated from Fusobacterium necrophorum or variant thereof.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences (preferably protein encoding sequences) that which naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated Adhesin nucleic acid molecule can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences have a common structural domain and/orcommon functional activity. For example, amino acid or nucleotidesequences which contain a common structural domain having about 50-99%or more identity are defined herein as sufficiently identical.Preferably, the amino acid or nucleotide sequence has about 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identity. Morepreferably, the amino acid or nucleotide sequence has about 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70% or more identity.More preferably, the sequence has about 60% identity.

The term “sample” refers to a cell, a population of cells, biologicalsamples, and subjects, such as mammalian subjects. The term “biologicalsample” refers to tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.

As used herein, “administering” is used in its broadest sense to meancontacting a subject with a composition of the invention.

As used herein, “subject” refers to a living organism having a centralnervous system. In particular, subjects include, but are not limited to,human subjects or patients and companion animals. Exemplary companionanimals may include domesticated mammals (e.g., dogs, cats, horses),mammals with significant commercial value (e.g., dairy cows, beefcattle, sporting animals), mammals with significant scientific values(e.g., captive or free specimens of endangered species), or mammalswhich otherwise have value. Suitable subjects also include: mice, rats,dogs, cats, ungulates such as cattle, swine, sheep, horses, and goats,lagomorphs such as rabbits and hares, other rodents, and primates suchas monkeys, chimps, and apes. In some embodiments, subjects may bediagnosed with a Fusobacterium infection, may be at risk forFusobacterium infection, or may be experiencing symptoms of aFusobacterium infection. Subjects may be of any age including new born,adolescence, adult, middle age, or elderly.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to increase to some beneficial degree, preferably toincrease by at least about 1 to 100 percent, more preferably by at leastabout 5 to 95 percent, and more preferably by at least 8 percent orhigher, healing or Fusobacterium cell death as compared to untreatedcontrols. An “effective amount” is a pharmaceutically-effective amountthat is intended to qualify the amount of an agent or compound, thatwhen administered to a subject, will achieve the goal of healing aninjury site, increasing Fusobacterium cell death, or otherwisebenefiting the recipient environment.

As used herein, the term “vector” refers to a nucleic acid sequencedesigned to be propagated and or transcribed upon exposure to a cellularenvironment, such as a cell lysate or a whole cell. A “gene therapyvector” refers to a nucleic acid vector that also carries functionalaspects for transfection into whole cells, with the intent of increasingexpression of one or more genes or proteins. In each case such vectorsusually contain a “vector propagation sequence” which is commonly anorigin of replication recognized by the cell to permit the propagationof the vector inside the cell. A wide range of nucleic acid vectors andgene therapy vectors are familiar to those skilled in the art.

The term “Adhesin agent” refers to any molecule capable of modulatingAdhesin activity, by increasing or decreasing the activity directly orindirectly. Exemplary Adhesin agents include, without limitation, acompound, drug, small molecule, peptide, immunogen, oligonucleotide,protein, antibody, and combinations thereof. Adhesin agents may besynthetic or naturally occurring. An Adhesin agent may be a moleculeidentified in a screening assay as described herein. Further, an Adhesinagent can be used to induce an immune response in a subject againstAdhesin.

The term “Adhesin composition” refers to any composition including atleast one Adhesin agent.

The term “Adhesin indicator” refers to any molecule capable of detectingthe presence of Adhesin. A suitable Adhesin indicator may be a compound,drug, small molecule, peptide, immunogen, oligonucleotide, protein,antibody, and combinations thereof.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a therapeutic agent of theinvention and a pharmaceutically acceptable carrier. As used herein,“pharmacologically effective amount,” “therapeutically effective amount”or simply “effective amount” refers to that amount of an agent effectiveto produce the intended pharmacological, therapeutic or preventiveresult. For example, if a given clinical treatment is consideredeffective when there is at least a 15% reduction in a measurableparameter associated with a disease or disorder, a therapeuticallyeffective amount of an agent for the treatment of that disorder ordisease is the amount necessary to effect at least a 15% reduction inthat parameter.

The term “pharmaceutically acceptable carrier” is used interchangeablywith “veterinary acceptable carrier” and both refer to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, andclactose, while corn starch and alginic acid are suitable disintegratingagents. Binding agents may include starch and gelatin, while thelubricating agent, if present, will generally be magnesium stearate,stearic acid or talc.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins eds.);Transcription and Translation, 1984 (Hames and Higgins eds.); AnimalCell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes,1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning;the series, Methods in Enzymology (Academic Press, Inc.); Gene TransferVectors for Mammalian cells, 1987 (J. H. Miller and M. P. Calos eds.,Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 andVol. 155 (Wu and Grossman, and Wu, eds., respectively).

EXAMPLES

The following examples are simply intended to further illustrate andexplain the present invention. The invention, therefore, should not belimited to any of the details in these examples.

Example 1: Materials and Methods

Bacterial Strains and Culturing.

EJG (bovine adrenal capillary endothelial cells) and CPAE (Calfpulmonary artery endothelial cells) were seeded into a 6 well plate at aconcentration of 1×10⁵ cell/ml and incubated in Eagle's MinimalEssential Medium (EMEM) containing 10% heat-inactivated FBS for 48 hoursto remove any effect of trypsinization on the eukaryotic surfaceproteins. Overnight cultures of FNN (strain 8L1) and FNF (strain B35),grown in pre-reduced, anerobically sterilized brain-heart infusion broth(PRAS-BHI) to reach mid-log phase (Absorbance₆₀₀ of 0.6), werecentrifuged to pellet cells, and then washed and resuspended in CO₂bubbled EMEM medium containing heat-inactivated Fetal Bovine Serum(FBS). The endothelial cells were washed thoroughly (4 times) withsterile PBS and treated with F. necrophorum at multiplicity of infectionof 100:1 for one hour at 37° C. in an anaerobic chamber. The cells werewashed again with sterile PBS, treated briefly with trypsin-versene toloosen the cells, and neutralized using CO₂ bubbled EMEM containingheat-inactivated FBS. This medium was serially-diluted in PRAS-BHI andplated quickly on blood agar plate pre-incubated in the anaerobicchamber to determine the bacterial concentration.

Fusobacterium necrophorum Clinical Isolates from Humans or Cattle.

Twenty-seven strains of Fusobacterium necrophorum (15 subsp. necrophorumand 12 subsp. funduliforme) were grown in PRAS-BHI broth, modifiedlactate broth and on blood agar plates were examined for the presence offimbriae, flagella, or capsule.

Polyclonal Antisera Raised Against OMPs (Outer Membrane Proteins).

Polyclonal antisera were raised in rabbits against the OMPs extractedfrom FNN (Fusobacterium necrophorum subsp. necrophorum) and FNF and(Fusobacterium necrophorum subsp. funduliforme)). Antibodies werepurified using Vivapure protein A spin columns (Satorius Stedim Biotech,Aubagne Cedex, France).

Extraction of Outer Membrane Proteins.

The outer membrane proteins from F. necrophorum subsp. necrophorumstrain 8L1 were isolated according to the method described by Osburn andMunson (1974) with slight modifications. Briefly, cultures were grown in1 liter of PRAS-BHI broth for 12 to 14 hours. The cells were pelleted bycentrifugation, resuspended in 20 ml of cold 0.75M sucrose-10 mM Trisbuffer at pH 7.8 with lysozyme (2 mg/ml of cell suspension) andincubated on ice for 20 minutes. Formation of spheroplasts was achievedby diluting the suspension with two volumes of cold 1.5 mM EDTA atconstant rate of delivery. The spheroplasts were lysed byultrasonication in an ice-water bath with a 3-mm microtip at 20-W outputpulse setting. The cell debris was removed by centrifugation at 1,200 gfor 15 minutes at 4° C. The supernatant was then centrifuged at 65,000rpm at 2-4° C. for 2 hours, and the supernatant was discarded. Thepellet was resuspended in a small volume of cold 0.25 M sucrose-3.3 mMTris-1 mM EDTA, pH 7.8 (STE buffer) and the volume was adjusted to thatof the original sonicate suspension and the ultracentrifugation wasrepeated at 60-65,000 rpm at 2-4° C. for 2 hours. The pellet wasresuspended in 2 ml of 20 mg/ml TRITON® X-100 detergent and 10 ml of STEbuffer and incubated at room temperature for 45 minutes to dissolve theinner membrane. The suspension was further centrifuged at 37,000 rpm for2 hours at 2-4° C. The pellet was collected in cold STE buffer andstored at −80° C. until use.

Culture of Endothelial Cells.

Bovine adrenal gland capillary endothelial (EJG cells) cell line wasused for this study. Cells were grown in EMEM medium with 10% fetal calfserum and 1% antibiotic solution of streptomycin and penicillin. Themedium was changed every 3-4 days until the cells got monolayered. Thecells were trypsinized when monolayered and subcultured to maintain thecell line for use in experiments.

Attachment of Fusobacterium necrophorum Subsp. Necrophorum toKarnovsky's Fixed Bovine Endothelial Cells.

The EJG were seeded into a 6 well plate at a concentration of 1×10⁵cell/ml and incubated in EMEM medium containing 10% FBS for 48 h toremove any effect of trypsinization on the eukaryotic surface proteins.The cells were then fixed with Karnovsky's fixative for 10 minutes,followed by washing with sterile PBS for 3-4 times. Overnight culturesof F. necrophorum subsp. necrophorum (strain 8L1) grown in pre-reduced,anaerobically sterilized brain-heart infusion broth (PRAS-BHI) to reachmid-log phase (Absorbance₆₀₀ of 0.6). Then, 1 ml of mid-log phase grownbacteria were inoculated on to the fixed cells and incubated at 37° C.for 1 hour. After incubation, the cells were washed vigorously withsterile PBS for 3-4 times and the bound bacterial were observed inmicroscope.

Isolation of High Affinity Binding OMP, FncA (40 kDa Adhesin).

The EJG cells were plated on 6-well plates and incubated for 48 hours toallow recovery of surface proteins (following trypsin treatment duringcell culture splitting). The cells were fixed in modified Karnovsky'sfixative and then incubated overnight with purified OMPs from subsp.necrophorum strain 8L1. After incubation, the unbound fraction wasremoved from the cells followed by washing of the cells with PBS twicefollowed by two washes each with increasing strengths of buffers: PBScontaining 0.1% NP-40, modified RIPA (25 mM Tris*HCl pH 7.6, 150 mMNaCl, 1% NP-40, and 1% sodium deoxycholate) and finally the tightlybound OMP was collected by washing with SDS sample buffer (62.5 mMTris-HCl pH 6.8, 25% Glycerol and 10% SDS). Each of the washes wasconcentrated to equal volume (10 times) and separated on a SDS-PAGE gel.The EJG cells not incubated with OMP served as negative control.

Protein Sequencing.

The purified protein in SDS-PAGE buffer was run on 4% stacking and 10%separation gel after overnight polymerization to avoid and N-terminalblockage. The protein was transferred to a PVDF membrane overnight byelectroblotting, stained with coomassie blue staining followed bydestaining. The stained PVDF membrane showing the purified OMP (FncA)was rinsed in distilled water for 4 hours while changing the waterseveral times. Finally, the PVDF membrane containing the protein wasdried between Whatman No. 1 filter paper and protein was excised to besent on dry ice to sequencing facility at Iowa State University (Ames,Iowa). The N-terminal protein sequencing was carried out with 494Procise Protein/peptide Sequencer/140C Analyzer using Edman degradationmethod (Edman, P et al. 1950). The identified amino acid sequence (SEQID NO: 2) was blasted in Pubmed database (on the internet atncbi.nlm.nih.gov/pubmed/) and investigated for similar proteins presentin other closely related bacterial species.

PCR Analysis of Different Strains of Subsp. Necrophorum, Subsp.Funduliforme and Human Strains of Fusobacterium necrophorum.

The presence of the fncA gene was investigated in different strains ofsubsp. necrophorum, subsp. funduliforme and human strains. DNA fromdifferent strains were isolated and subjected for PCR analysis usingprimers based on the DNA sequence of homologous proteins present inother closely related bacterium Fusobacterium nucleatum. The PCRconditions were as follows: 1 cycle 94° C. for 3 minutes followed by 35cycles of denaturation at 94° C. for 1 minute, annealing at Tm−5° C. for30 seconds, and extension at 72° C. (1 minute per kb of expectedproduct).

Detection of Fibronectin on EJG Cell Culture.

The cells were grown in 24 well plates and fixed with 10% methanol for10 minutes followed by blocking with 2% non fat dry milk in PBST forhalf an hour. The cells were then incubated with DAPI and mouseanti-fibronectin antibody followed by washing and incubation withrhodamin conjugated Affinipure goat anti-mouse IgG antibody. The stainedcells were visualized using immunofluorescence microscope (Nikon EclipseTE-2000-U).

Far-Western Analysis of FncA with Fibronectin.

Bovine fibronectin (1 mg) was dissolved 1 ml of PBS (final concentration1 μg/μl) and 250 μl was incubated with 250 μl of 10 mM EZ-LinkSulfo-NHS-LC-Biotin solution in PBS for 30 minutes at room temperaturewith constant shaking. The excess of biotin was neutralized by adding500 μl of 1 M Tris-HCl and incubating for additional 15 minutes. Theneutralized mixture was further washed 3-4 times with 5 ml PBS using 100kDa filter and concentrated to a final volume of 200 μl. Thebiotinylated sample was then incubated with FncA protein blotted onnitrocellulose membrane for 2 hours at room temperature in PBST(PBS+0.05% TWEEN® 20 detergent) followed by washing with PBST threetimes for 10 minutes each. The biotinylated fibronectin bound to thepurified FncA was detected using Avidin-HRP conjugate. The proteinincubated only with Avidin-HRP served as control. To further avoid anysuspicion of biotin directly binding to the purified FncA protein, amodified approach was taken where the fibronectin was directly incubatedwith the blotted FncA for 2 hours followed by incubation withmouse-Anti-Fibronectin antibody for 2 hours. After incubation, theblotted membrane was washed with PBST and incubated with anti-mouse IgGAb conjugated with alkaline phosphatase and the color was developedusing BCIP/NBT substrate.

Inhibition Assay.

The outer membrane proteins of subsp. necrophorum strain 8L1 (400 μg)was incubated in each well of karnovsky's fixed EJG cells grown on 6well plate overnight and washed with PBS twice to remove unboundproteins followed by two washing of PBS+0.1% NP-40 so as to leave onlyFncA OMP bound to the fixed cells. Subsp. necrophorum strain 8L1 weregrown to 0.6 OD in PRAS-BHI medium by reinoculating overnight grownculture and 1 ml of the medium containing the bacterium were inoculatedto each well of Karnovsky's fixed EJG cells. The bacteria were incubatedwith fixed cells for 1 hour followed by washing with PBS vigorously for3-4 times to remove unbound bacteria. The bound bacteria to the EJGcells were removed by quickly scraping the wells after adding 500 μl oftrypsin and inactivating the trypsin with 1.5 ml of CO₂ bubbled EMEMmedium containing 10% inactivated FBS. A 100 μl of the extracted mediumcontaining the bacteria was serially diluted in PRAS-BHI and plated onblood agar plate for enumeration.

Cloning, Sequencing and Protein Expression of FncA from Subsp.Necrophorum.

pET22b+ vector was used to clone and express the fncA gene from subsp.necrophorum strain 8L1 DNA. The forward primer5′-CGGGATCCAGAAGTTATGCCTGCACC-3′ (SEQ ID NO: 6) had a BamHI site andreverse primer 5′-GGTGGCGGCCGCGAAAGTAACTTTCATACCAGC-3′ (SEQ ID NO: 7)had NotI site for directional cloning. The enzymes for directionalcloning were selected after digesting the initial PCR product that wereamplified using primers based on homologous gene sequence as mentionedbefore with different digestive enzymes listed on multiple cloning siteof pET22b+ vector and the enzymes that did not digest the product wereconsidered suitable for cloning. The amplified product was cloned inpET22+ vector in-frame which contains a PelB leader peptide in theN-terminal and a histidine tag on the C-terminal of the cloning site.PelB leader peptide helps the cloned protein to be secreted insupernatant. The cloned vector was transformed in BL21(DE3) cells andthe right clones were verified using colony PCR. A few of the clonesthat were positive in colony PCR were subjected for protein expressionusing IPTG by inducing the cells for 24 hours after inoculating theovernight grown clones in fresh LB medium and a few of those that showedprotein expression, the sequence of the cloned DNA was determined by DNAsequencing. The protein sequence of the DNA cloned was deduced form theDNA sequencing result and further analyzed for its similarity withhomologous proteins present in the database.

Western Blot Analysis of Recombinantly Expressed FncA with Bovine SerumChallenged with F. necrophorum Subsp. Necrophorum Strain 8L1.

The supernatant of E. coli BL21(DE3) cells expressing the recombinantFncA protein was subjected to SDS-PAGE analysis and blotted onnitrocellulose membrane. The blotted proteins were detected using serumfrom steers that were challenged with subsp. necrophorum strain 8L1 asprimary antibody and goat anti-bovine IgG antibody conjugated withalkaline phosphatase as secondary antibody. The color was developedusing BCIP/NBT substrate.

Bacterial Preparations.

Fusobacterium necrophorum subsp. necrophorum strain 8L1 previouslyisolated from liver abscess of cattle (Narayanan et al., 1997) andstored at −80° C. was streaked onto blood-agar plates (Remel, ThermoFisher Scientific, Lenexa, Kans.) in an anaerobic glove box (FormaScientific, Marietta, Ohio; model 1024). The bacterial colonies wereGram-stained and their biochemical characteristics were determined usinga RapID ANA II kit (Remel) in order to confirm the identity and purityof F. necrophorum. After the bacterial identity was confirmed, 10 mlpre-reduced anaerobic sterilized brain heart infusion broth (PRAS-BHI[Tan et al., 1992]; Becton-Dickinson, and Company, Franklin Lakes, N.J.)tubes were inoculated with single colonies and grown overnight at 39° C.to achieve starter cultures.

Animal Assignment.

At 7 weeks of age, 54 five-week-old female CF1 mice (multipurpose mousemodel for infectious disease) were purchased and sorted three per cage.Two weeks later, the mice were assigned into six groups at random (9mice per group; Table 1). Groups 1 and 2 were assigned as no-vaccinecontrols (phosphate buffered saline [PBS]+TITERMAX® Classic adjuvant(adjuvant) [Stratech Scientific Ltd. Suffolk, U.K]), group 3 wasassigned for vaccine candidate 1 (40 kDa adhesin+adjuvant), group 4 wasassigned for vaccine candidate 2 (recombinant leukotoxin+adjuvant),group 5 was assigned for vaccine candidates 1+2, and group 6 wasassigned for leukotoxoid vaccine (a formalin-treated culture supernatantof F. necrophorum strain A25+adjuvant).

TABLE 1 Vaccination and Challenge Protocol. Group 1 Group 2 Group 3Group 4 Group 5 Group 6 Day −14 Arrival of 90 mice, randomization andseparation into six groups Day 0 PBS + PBS + Vaccine 1 Vaccine 2 VaccineLeukotoxoid adjuvant adjuvant (40 kDa (recombinant 1 + 2 (40 kDa(formalin adhesion + leukotoxin + adhesion + treated A25 adjuvant)adjuvant) recombinant supernatant + leukotoxin) adjuvant) Day 14 PBS +PBS + Vaccine 1 Vaccine 2 Vaccine Leukotoxoid adjuvant adjuvant (40 kDa(recombinant 1 + 2 (40 kDa (formalin adhesion + leukotoxin + adhesion +treated A25 adjuvant) adjuvant) recombinant supernatant + leukotoxin)adjuvant) Day 21 PBS + PBS + Vaccine 1 Vaccine 2 Vaccine Leukotoxoidadjuvant adjuvant (40 kDa (recombinant 1 + 2 (40 kDa (formalinadhesion + leukotoxin + adhesion + treated A25 adjuvant) adjuvant)recombinant supernatant + leukotoxin) adjuvant) Day 42 PBS onlyIntraperitoneal challenge with F. necrophorum (3.6 × 10⁶) Day 46Euthanasia, organ harvesting, and plating

Example 2: Fusobacterium necrophorum Subspecies Necrophorum Binds toBovine Endothelial Cells with High Affinity

To determine if host cell attachment is part of the pathologic processof Fusobacterium necrophorum, endothelial binding assays were conducted.In particular, the binding of FNN and FNF strains to EJG cells wasanalyzed. As detailed in Example 1, two endothelial cell lines, EJG andCPAE, were incubated with either FNN or FNF bacteria and analyzed forbinding. The EJG cell line consistently exhibited more binding comparedto the CPAE cell line, and was therefore used for further studies. Also,the FNN strain was found to bind with higher affinity to EJG cellscompared to FNF (FIG. 1 and FIG. 9). Similar binding experiments werealso performed using EJG cells treated with modified Karnovsky'sfixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylatebuffer pH 7.4) for 10 minutes. No significant differences were observedbetween fixed and unfixed cells in the ability of FNN or FNF bacteria toattach to the cells.

The binding of gram-negative bacteria to eukaryotic host cells may bemediated by several different mechanisms including throughoutermemberane capsule, fimbria, or flagella structures. To determine ifthe binding of FNN or FNF is mediated by these outer membranestructures, the binding of EJG was further analyzed by examiningFusobacterium necrophorum clinical isolates from humans or cattle.Bacterial cultures of twenty-seven strains of Fusobacterium necrophorum(15 subsp. necrophorum and 12 subsp. funduliforme) were delicatelywashed and negatively stained with 1% phosphotungstic acid. When viewedunder transmission or scanning electron microscope, no fimbria, flagellaor capsular structures were present in any of the bacterial cultures ofthe twenty-seven strains (FIGS. 2A and 2C). These results were furtherconfirmed by transmission electron microscopy of bacterial culturesprocessed routinely by post fixation in 1% osmium tetraoxide andpositive staining with uranyl acetate and lead citrate (FIGS. 2B and2D). None of the twenty-seven examined strains had surface appendagessuch as flagella or fimbria. All F. necrophorum strains appeared underEM as short or long rods frequently arranged as short-chains and hadtypical Gram-negative cell wall structures (FIGS. 2C and 2D).

To further investigate the host cell binding, or attachment, mechanismof FNN and FNF, outer membrane proteins of each species were isolatedand then compared. Specifically, the outer membrane proteins (OMPs) ofFNN and FNF isolated from cattle were extracted using the methoddescribed by Osburn and Munson (1974) with slight modifications (See,Example 1 for details). Briefly, differences in OMP profiles of FNN andFNF were evaluated by SDS-PAGE and western blot analyses to identifyproteins that may be related to the higher virulence of FNF. The outermembrane proteins were extracted and separated in SDS-PAGE, staineddirectly to visualize the bands and then transferred to nitrocellulosemembrane for detection with the sera from slaughtered cattle with liverabscesses. The SDS-PAGE revealed that FNN strains had major proteinbands which were absent in FNF strains. These proteins may contributetowards higher pathogenicity of FNN. As shown in FIG. 3, there wereconsiderable differences in the OMP profiles of FNN and FNF isolatedfrom cattle.

It was further observed that trypsin treatment of FNN reduced binding toEJG cells. Treatment of FNN for 10 minutes in EMEM containing trypsin,followed by thorough washing, significantly reduced its ability to bindto EJG cells, indicating that proteins on the surface of bacterium playan important role in binding.

Purified OMPs and polyclonal antisera raised against OMPs reducedbinding of FNN to EJG cells. Fixed EJG cells were incubated with OMPpurified from FNN before performing the binding assays. There was asignificant reduction in binding of FNN (FIG. 4). Polyclonal antiserawere raised in rabbits against the OMPs extracted from FNN and FNF.Antibodies were purified using Vivapure protein A spin columns. FNNcultures pretreated with antiserum against OMPs of FNN reduced theirbinding to EJG cells, whereas the antiserum against OMPs of FNN did nothave significant inhibitory effect on binding (FIG. 4). Antiserumagainst OMPs of FNF significantly reduced binding of FNF strains (butnot FNN strains) to EJG cells. This subspecies-specific inhibition ofbinding suggests differences between FNF and FNN in the OMPs involved intheir binding to EJG cells. The attachment of FNN to EJG cells (FIG. 4,#1), was reduced significantly when pretreated with polyclonal antiseraraised against OMP of FNN (FIG. 4, #2). The attachment was not reducedwhen pretreated with polyclonal antisera raised against OMP of FNF (FIG.4, #3), but was reduced significantly when EJG cells were pretreatedwith subsp. necrophorum OMPs (FIG. 4, #4) or with 40 kDa adhesin (FIG.4, #5). FNN cultures pretreated with antiserum against OMPs of FNNreduced their binding to EJG cells, whereas the antiserum against OMPsof FNN did not have significant inhibitory effect on binding (FIG. 4).Antiserum against OMPs of FNF significantly reduced binding of FNFstrains (but not FNN strains) to EJG cells. This subspecies-specificinhibition of binding suggests differences between FNF and FNN in theOMPs involved in their binding to EJG cells.

Example 3: Putative Adhesins of Bovine F. necrophorum SubspeciesNecrophorum

Putative adhesins of bovine FNN isolates were identified using themethod in Example 1. Briefly, the EJG cells were plated on 6-well platesand incubated for 48 hours to allow recovery of surface proteins(following trypsin treatment during cell culture splitting). The cellswere fixed in modified Karnovsky's fixative and then incubated overnightwith purified OMPs from FNF. The cells were first washed with PBSfollowed by two washes each with increasing strengths of buffers: (1)PBS containing 0.1% NP-40; (2) modified RIPA (25 mM Tris*HCl pH 7.6, 150mM NaCl, 1% NP-40, and 1% sodium deoxycholate); and, (3) final wash withSDS sample buffer (62.5 mM Tris-HCl pH 6.8, 25% Glycerol and 10% SDS).Each of the washes was concentrated 10 times and separated on a SDS-PAGEgel (FIG. 5). As shown in FIG. 10, when the OMP were incubated overnightto fixed EJG cells and the unbound fraction was removed (lane 2)followed by washing with PBS (lane 3) and detergents with increasingstringency, no protein could be detected in washing with PBS+0.1% NP-40(lane 5 as treated cells and 6 as control cells), or modified RIPA (25mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, and 1% sodium deoxycholate)(lane 7 as treated cells and 8 as control cells). The final wash withSDS-PAGE buffer containing 10% SDS could only detach the tightly boundOMP (lane 10 as treated cells and lane 11 as control cells) which wasaround 40 kDa in size.

The concentrated final wash preparation revealed a 40 kDa protein ingels stained with colloidal Coomassie blue. The concentrated final washpreparation was also blotted on to a nitrocellulose membrane. Westernanalyses of this blot using polyclonal antisera raised against OMPs ofFNN or FNF detected the 40 kDa protein and two additional proteins:sizes 70 kDa and 19 kDa proteins (FIG. 5). Far-western analysis of theblot revealed that bovine fibronectin bind with high affinity to the 40kDa protein (FIG. 5). Briefly, for the far-western analysis, bovinefibronectin was biotinylated, and was used to hybridize the blotcontaining putative adhesins, and horseradish peroxidase tagged toavidin was used for detection.

The OMPs from FNF were used in a competitive binding assay to evaluateany potential role in host cell attachment the OMPs may have. It wasobserved that adding the purified OMPs reduced binding of FNN to EJGcells due to binding competition, which verified the function of theputative adhesins isolated. The fixed EJG cells were treated withpurified OMPs. The wells were washed twice with PBS, followed by twowashes in PBS+0.1% NP-40. In binding assays, there was a significantreduction in the number of FNN that attached to EJG cells, compared toEJG cells untreated with OMPs (FIG. 4, #4 and #5).

Example 4: Protein Analysis and In Vitro Expression of the Putative 40kDa Adhesin

The 40 kDa adhesin has not been shown previously to be present in F.necrophorum or implicated as a protein that facilitates attachment tohost cell surfaces. To further characterize the 40 kDa adhesion, the 40kDa protein was sequenced and analyzed using in vitro expressionexperiments. The piece of SDS-PAGE gel containing the 40 kDa proteinfrom Example 2 was cut and submitted for protein microsequencing. TheN-terminal amino acid sequence (Iowa State Univ. sequencing facility) ofthis protein was K-E-V-M-P-A-P-M-P-E-D/E-E (SEQ ID NO: 3, 4, and 5).Blast-P sequence analysis revealed that this protein has 96% homology toa 40 kDa OMP of F. nucleatum and F. periodonticum (FomA) and F. varium.Sequence analysis also revealed that the proteins in other fusobacteriahave a 20-aa signal peptide that is cleaved upon its expression into theperiplasm. Protein modeling shows the structural features of 40 kDaAdhesin (FIGS. 16, 17, and 18). Degenerate primers were synthesizedbased on the N-terminal protein sequence and the DNA sequence of F.nucleatum published in Genbank X72582.1. The PCR amplified a fragment of1.25 kb with cattle isolates of subsp. necrophorum, and a slightlysmaller 1.15 kb fragment with cattle isolates of subsp. funduliforme(FIG. 6). Interestingly, these primers amplified a 1.25 kb fragment whenthe DNA from human strains was used as templates. Nucleotide sequenceanalysis of the PCR products revealed that the gene encoding this 40 kDaOMP in two subspecies had only 42% homology between them and thesequence falls out of frame after 61 amino acids and encounters a stopcodon after a total of 70 amino acids in FNF.

In order to clone and express the 40 kDa adhesion (SEQ ID NO: 1 and 2),primers were designed with restriction sites compatible for cloning theinto pET22b+ vector, which encodes a PelB leader peptide to transportthe expressed protein into periplasm of the E. coli. The cloned vectorwas transformed into E. coli BL21 (DE3) and induced with IPTG forexpressing the proteins. A peptide of approximately 40 kDa was presentin the supernatant after a six-hour induction, and this protein wasdetected using anti-5× histidine tag antibody, rabbit polyclonalantiserum against OMP of FNN, and serum collected from cattleexperimentally challenged with FNN to induce liver abscesses (FIG. 7).This protein has also been cloned, expressed and purified in a pET45b+vector where the protein is expressed under the control of the T7promoter and IPTG induction leads to sequestration of the protein in E.coli BL21 (DE3) inclusion bodies. The protein expression was confirmedby western blot assays using antibody for the 6× histidine tag on theN-terminus of the recombinant adhesion.

To further investigate if the identified 40 kDa adhesin is present indifferent strains of subsp. necrophorum, subsp. funduliforme, and humanstrains of Fusobacterium necrophorum, PCR analysis was carried out whichshowed that the gene is present in both subsp. necrophorum and subsp.funduliforme (FIG. 11A). PCR analysis of human strains of F. necrophorumalso showed the presence of this gene in all the four strains tested(FIG. 11B).

Antibodies directed against 40 kDa adhesion reduced the attachment ofFNN to EJG cells. The recombinant 40 kDa protein purified by IMACchromatography on nickel-chelation columns under denaturing conditions(with 6M urea) was used for vaccinating rabbits. FNN cultures pretreatedwith post-vaccination serum had significantly lower binding to EJG cellscompared to the pre-vaccination serum (FIG. 16).

Fluorescence microscopy was applied to observe the binding activity ofthe 40 kDa protein and it revealed that the 40 kDa protein binds to thesurface of EJG cells. The 40 kDa protein recovered from the SDS-PAGE gelwas concentrated using Microcon YM-3 columns. Fixed EJG cells wereincubated overnight with this protein preparation. Polyclonal antiserumagainst OMP of FNN was used as a primary antibody and goat anti-rabbitantibody tagged to rhodamine was used as the secondary antibody, and thecells were visualized using confocal microscopy (Nikon Eclipse TE2000-Econfocal microscope). The 40 kDa protein bound tightly to the surface ofEJG cells (FIG. 8). To check the specificity of the secondaryantibodies, incubation with primary antibodies was omitted in negativecontrols.

Immunohistochemistry studies showed that the EJG cells were producingfibronectin in the cell culture as shown in FIG. 12. Many outer membraneproteins of different Gram negative bacteria have been shown to bindfibronectin. The far-western analysis using biotinylated bovinefibronectin showed that the identified 40 kDa protein binds with highaffinity to the fibronectin (FIG. 13). Further analysis usingnon-biotinylated bovine fibronectin confirmed that the binding seen inthe far-western analysis is only due to actual binding of the bovinefibronectin to the identified 40 kDa protein and is not due to anyexcess unneutralized biotin left in the experimental step (FIG. 14).

Example 5: Fusobacterium Adhesin Vaccine

A vaccine was prepared using the 40 kDa Adhesin protein FncA evaluatedin Example 4. The E. coli clones that express recombinant 40 kDa Adhesinwere grown in 10 ml Luria-Bertani broth with 50 μg/ml ampicillinovernight. The following day 100 ml LB broth was inoculated with 5 ml ofthe overnight culture and grown to OD of 0.4 before induction withisopropylthiogalactopyranoside (IPTG). Induction time varied from 1.5hours to 2 hours depending on the protein expression profiles. Afterinduction, bacteria were pelleted at 5,000 rpm for 10 minutes and theproteins were purified under denaturing conditions using 6M urea. Thepolypeptides were dialyzed with Microcon YM-10 filters (Millipore) toexclude proteins smaller than 10 kDa. Protein concentrations werechecked using Bradford method. After purification, all proteins wereelectrophoresed on a SDS-PAGE gel and stained with Coomassie Blue toensure correct size of the protein. Correct protein size was alsoconfirmed by western blot using Penta His antibodies. Immunepreparations for each dose of Adhesin vaccine contained 30 μg of theFncA 40 kDa protein mixed with TITERMAX® Classic adjuvant (adjuvant)(Stratech Scientific Ltd.).

Example 6: Fusobacterium Leukotoxin and Recombinant Leukotoxin Vaccine

The E. coli clones that express recombinant leukotoxin were grown in 10ml Luria-Bertani broth with 50 μg/ml ampicillin overnight. The followingday 100 ml LB broth was inoculated with 5 ml of the overnight cultureand grown to OD of 0.4 before induction withisopropylthiogalactopyranoside (IPTG). Induction time varied from 1.5hours to 2 hours depending on the protein expression profiles. Afterinduction, bacteria were pelleted at 5,000 rpm for 10 minutes and theproteins were purified under denaturing conditions using 6M urea. Thepolypeptides were dialyzed with Microcon YM-10 filters (Millipore) toexclude proteins smaller than 10 kDa. Protein concentrations werechecked using Bradford method. After purification, all proteins wereelectrophoresed on a SDS-PAGE gel and stained with Coomassie Blue toensure correct size of the protein. Correct protein size was alsoconfirmed by western blot using Penta His antibodies. Immunepreparations for each dose of recombinant leukotoxin vaccine contained30 μg of recombinant leukotoxin protein mixed with TITERMAX® Classicadjuvant (adjuvant) (Stratech Scientific Ltd.). The no-vaccine controlsreceived PBS mixed with the adjuvant. The leukotoxoid vaccine for group6 was prepared by streaking frozen F. necrophorum subsp. necrophorumstrain A25 strain on blood-agar plates. Following growth, individualcolonies were inoculated in RAS-BHI broth and grown overnight. Overnightstarter cultures were then used to inoculate fresh PRAS-BHI broth andincubated to reach an OD600 of 0.7 (approximately 7 hours). The bacteriawere pelleted, and the supernatant was treated with polymyxin B sulfate(50 μg/ml)+0.3% formalin. All vaccines were administered viaintramuscular injection into the quadriceps muscle of mice on days 0,14, and 21.

Example 7: Analysis of Fusobacterium necrophorum 40 kDa Adhesin Vaccineand Leukotoxin Vaccines

Vaccine for inducing an immune response in a subject to 40 kDa Adhesin(40 kDa Adhesin Vaccine) was prepared as described in Example 5. Toevaluate the efficacy of the 40 kDa Adhesin vaccine, CF1 mice werechallenged with F. necrophorum after receiving a vaccination of 40 kDaAdhesin vaccine, leukotoxin vaccine, or sterile PBS (as a control). Themice were assigned into six groups at random (9 mice per group; Table1). Groups 1 and 2 were assigned as no-vaccine controls (PBS+adjuvant),group 3 was assigned for 40 kDa adhesion+adjuvant vaccine, group 4 wasassigned for recombinant leukotoxin+adjuvant vaccine, group 5 wasassigned for 40 kDa+recombinant leukotoxin+adjuvant vaccine, group 6 wasassigned for leukotoxoid vaccine (formalin-treated culture supernatantof F. necrophorum strain A25+adjuvant).

Blood samples were collected in 1.7 ml micro-centrifuge tubes containingheparin sodium salt, on days 0, 14, 21, 42 and 46 from the submandibularvein using goldenrod animal lancets. The volume of blood collectedaccounted for less than 1% of the total weight of the mouse. Plasma wasextracted from the blood samples by centrifuging the blood samples at4,000 rpm for 10 minutes, and was stored at −20° C. until furtheranalysis. Western-blot analyses with serial dilutions of plasma wereperformed to determine antibody titers against specific antigens(adhesin and leukotoxin). Total outer membrane proteins of F.necrophorum subsp. necrophorum strain 8L1 were purified using standardprotocols and were used in western-blots to determine the anti-adhesintiters. Culture supernatant of F. necrophorum subsp. necrophorum strainA25 (OD600=0.7) was concentrated 80-fold and used as antigen inwestern-blots to test antileukotoxin antibody titers. Plasma dilutionsof 1:200, 1:1000, 1:5000, 1:10,000, and 1:20,000 were made in blockingbuffer (2% blocking-grade non-fat dry milk in phosphate buffered salinewith 0.05% TWEEN® 20 detergent [PBST]). The dilutions were loaded ontothe mini-protean II multiscreen device for 4-10 hours on a rocker at 4°C. The blots were then washed three times with PBST. After washing, 10ml of blocking buffer containing 5 μl Anti-Mouse IgG produced in goatand conjugated with alkaline phosphatase was added to the blot androcked for 1.5 hours at room temperature. The blots were then washedwith PBST and developed with BCIP/NBT premixed solution.

On day 42, groups 2 through 6 were challenged by intraperitonealinjection with F. necrophorum strain 8L1. The inoculum was prepared asfollows: fresh pre-warmed PRAS-BHI broth tubes were inoculated with 300μl or 600 μl of the overnight starter culture and grown to OD600 of 0.7.The resultant culture was diluted twenty times in PRAS-BHI broth andeach mouse in groups 2 through 6 received a 400 ul intra-peritoneally(3.6×10⁶ total bacteria).

After the challenge, the mice were monitored every 4 hours during thelight cycle to observe signs of clinical illness. Clinical signs ofillness include ruffled coat, dehydration, and lethargy. The morbiditywas scored based on the following criteria (0=no clinical symptoms,1=mild ruffled coat, 2=ruffled coat, 3=ruffled coat+lethargy, 4=severedehydration/lethargy/severe ruffled coat, and 5=no response to externalstimuli).

All mice were euthanized on day 46. Organs collected at harvestingincluded liver, heart, lung, spleen, and brain. The liver, lungs, andspleen were each homogenized in 9 times the volume of modified lactatebroth for most probable number analysis. Ten-fold serial dilutions 10-1through 10-8 in modified lactate medium were attained using 96 wellplates. The homogenated samples were also streaked on blood agar platesand incubated both anaerobically and aerobically to test for any otherbacteria that may be present in the samples. Gram-staining and RapID AnaII test were also performed on each sample to confirm the identity of F.necrophorum. Brain, heart, and sections of lung, liver, and spleen werestored in 10% formalin for further microscopic analysis.

Serum antibody titers were analyzed using Graphpad Prism 5.03. Theantibody titers, morbidity, liver abscess formation, and isolation of F.necrophorum were evaluated. Any probability (P) values <0.05 wereconsidered significant. Repeated measures ANOVA test was performed andthe post test data was analyzed by Tukey multiple comparison tests. Datawas also run through a Friedman test with the post data analyses inDunn's multiple comparison tests.

Immunogenicity of vaccine preparations Mice in group three (40 kDaadhesin) exhibited significant rise in antibody titers (average 4444;Table 2) against F. necrophorum 8L1 adhesin by day 21 when compared today 0 (P<0.001), and to mice in group one (no vaccine no infection;P<0.001). Mice in group six (leukotoxoid vaccine) showed significantantibody production against F. necrophorum A25 supernatant leukotoxin byday 46 compared to day 0 (P<0.05), and to mice in group one (P<0.0197).

TABLE 2 Antibody titer values against native adhesion (Western blot).Treatment Day Day Day Day Day 46 Groups 0 13 21 42 (necropsy) No Vaccine(PBS) 0^(a) 0^(a) 0^(a) 0^(a) 0^(a) and No Challenge No Vaccine (PBS)0^(a) 0^(a) 0^(a) 0^(a) 0^(a) and Challenge 40 kDa Adhesin 0^(a) 0^(a)4444.4^(b)* 5000^(b)* 20,000^(b)** and Challenge Recombinant 0^(a) 0^(a)1000^(a) 1000^(a) 1000^(a) Leukotoxin and Challenge 40 kDa Adhesin +0^(a) 0^(a) 1444.4^(a) 1777.7^(b) 2555.5^(b) Recombinant Leukotoxin andChallenge Leukotoxoid 0^(a) 0^(a) 0^(a) 0^(a) 0^(a) (culturesupernatant) and Challenge Within a row, mean titers that do not havethe same superscript letters differ significantly (P < 0.05). *P < 0.05(different from the PBS control group). **P < 0.001 (different from thePBS control group).

The 40 kDa vaccine was highly immunogenic and was better at inducingantibodies than the leukotoxoid vaccine, for example, in group 6(leukotoxoid vaccine) the anti-leukotoxin antibody titer was 500 on day46 compared to an anti-40 kDa adhesin antibody titer of 20,000 on day 46in mice belonging to group 3 (40 kDa adhesin). Also, the 40 kDa vaccineelicited a quicker significant antibody response against adhesin by day21 (just after one booster dose) compared to the leukotoxoid vaccinewhich had a significant antibody response to leukotoxin only by day 46(after two boosters; Table 3).

TABLE 3 Antibody titer against leukotoxin. Titer values Day Day Day DayDay 46 (9 in each group) 0 14 21 42 (necropsy) No Vaccine (PBS) 0^(a)0^(a) 0^(a) 0^(a) 0^(a) and No Challenge No Vaccine (PBS) 0^(a) 0^(a)0^(a) 0^(a) 0^(a) and Challenge 40 kDa Adhesin 0^(a) 0^(a) 0^(a) 0^(a)0^(a) and Challenge Recombinant 0^(a) 0^(a) 0^(a) 0^(a) 0^(a) Leukotoxinand Challenge 40 kDa Adhesin + 0^(a) 0^(a) 0^(a) 0^(a) 0^(a) RecombinantLeukotoxin and Challenge Leukotoxoid 0^(a) 0^(a) 0^(a) 188.8^(a)400^(b)* (culture supernatant) and Challenge Within a row, mean titersthat do not have the same superscript letters differ significantly (P <0.05). *P < 0.05 (different from the PBS control group).

Over the period of observation (all five days post-infection), mice ingroup two (no vaccine control) had a significantly higher averagemorbidity score (2.33, P<0.05). Mice in other bacterial challengedgroups did not have any significant differences in morbidity rates(Table 4).

TABLE 4 Average morbidity scores of animals on Day 5 post challenge.Treatment Groups Average Morbidity Score 1 (No Vaccine [PBS] and NoChallenge) 0 2 (No Vaccine [PBS] and Challenge) 2.33* 3 (40 kDa Adhesinand Challenge) 1.33 4 (Recombinant Leukotoxin and Challenge) 1.78 5 (40kDA Adhesin + Recombinant 1.78 Leukotoxin and Challenge) 6 (Leukotoxoid(culture supernatant) 1.78 and Challenge) Scoring criteria: 0 = noclinical symptoms, 1 = mild ruffled coat, 2 = ruffled coat, 3 = ruffledcoat + lethargic, 4 = severe dehydration/lethargy/severe ruffled coat,and 5 = no response to external stimuli; *P < 0.05 (different from thePBS control group).

In group two (no-vaccine control), five of the nine mice had liverabscesses (FIG. 1; Table 5). In groups three (40 kDa adhesin vaccine)and five (40 kDa adhesin+recombinant leukotoxin) one of the nine miceper group had liver abscesses (both had a p value of 0.0665 compared togroup 2). Group six (leukotoxoid) and group four (recombinantleukotoxin) had the highest incidence of liver abscess, occurring inthree out of nine mice per group. Group one (no vaccine, no infectioncontrol), as expected, had no cases of liver abscess.

TABLE 5 Challenge Study: Liver abscess formation, isolation of F.necrophorum from liver, and isolation of F. necrophorum from liver,lung, and spleen (systemic infection). Isolation of Systemic Liverabscess F. necrophorum infection with formation from liver F.necrophorum (total of nine (total of nine (total of nine Group mice pergroup) mice per group) mice per group) 1 (No Vaccine, No 0 0 0 InfectionControl) 2 (No Vaccine 5^(a) 5^(a) 5^(a) Control) 3 (40 kDa Adhesin1^(a)* 4^(a) 3^(a) Vaccine) 4 (Recombinant 3^(a) 4^(a) 3^(a) LeukotoxinVaccine) 5 (40 kDA Adhesin + 1^(a)* 3^(a) 2^(a) Recombinant LeukotoxinVaccine) 6 (Leukotoxoid 3^(a) 4^(a) 3^(a) Vaccine) Within each column,means that have the same superscript letters are not significantlydifferent (P < 0.05); *P value of 0.0665 compared to group two.

In group two (no-vaccine control), F. necrophorum was isolated from theliver in five out of nine mice. In groups three (40 kDa adhesinvaccine), four (recombinant leukotoxin vaccine), and six (leukotoxoidvaccine), F. necrophorum was isolated from the liver in four out of ninemice in each group. Of the nine mice in group five (40 kDaadhesin+recombinant leukotoxin vaccine) F. necrophorum was isolated fromthe liver of three mice (Table 5). There was a significant correlationbetween livers carrying F. necrophorum and the presence of liverabscesses (p<0.01). As expected, F. necrophorum was not isolated fromany mouse from group one (no vaccine, no infection control).

Mice were considered to carry systemic infection if F. necrophorum wasisolated from the liver, lung, and spleen on anaerobic plates (dividedinto three sections; FIG. 20). Group one (no vaccine, no infectioncontrol) had no cases of systemic infection. Group two (no-vaccinecontrol) had the highest rate of F. necrophorum systemic infection,occurring in five out of nine mice. Group five (40 kDaadhesin+recombinant leukotoxin vaccine) had the lowest (two) cases ofsystemic infection. Groups three (40 kDa adhesin vaccine), four(recombinant leukotoxin vaccine), and six (leukotoxoid vaccine) each hadthree cases of systemic infection (Table 5).

In summary, these results show that the recombinant 40 kDa adhesin isimmunogenic and immunoprotective, and serves as an effective vaccine toprevent or control hepatic abscesses in subjects. Only two vaccinations(one initial dose and one booster dose) with the recombinant 40 kDaadhesin induced significant specific antibody response in mice. Thegroups that were vaccinated with recombinant 40 kDa adhesin by itself(group three) or in combination with recombinant leukotoxin (group 5)had the lowest incidences of liver abscesses (one out of nine mice pergroup) compared to the no-vaccine control group. Fewer mice in groupsthree and five had systemic infection with F. necrophorum on five-daypost-infection compared to the no-vaccine control group. F. necrophorumwas re-isolated from the liver of fewer mice in groups three and fivecompared to the no-vaccine control group. Also, Mice in group three [40kDa adhesin] exhibited lower incidence of liver abscesses, lessermorbidity, and a quicker antibody response than group six (leukotoxoidvaccine).

The invention illustratively disclosed herein suitably may be practicedin the absence of any element, which is not specifically disclosedherein. It is apparent to those skilled in the art, however, that manychanges, variations, modifications, other uses, and applications to themethod are possible, and also changes, variations, modifications, otheruses, and applications which do not depart from the spirit and scope ofthe invention are deemed to be covered by the invention, which islimited only by the claims which follow.

What is claimed is:
 1. An immunogenic composition comprising: a. anisolated Fusobacterium necrophorum peptide having a sequence selectedfrom the group consisting of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No.5; and, b. a pharmaceutically acceptable carrier comprising a componentselected from the group consisting of adjuvants, stabilizing agents,preservatives, antibacterial agents, antifungal agents, adsorptiondelaying agents, and any combination thereof.
 2. The immunogeniccomposition of claim 1, wherein the adjuvant is selected from the groupconsisting of surfactants, oil, mycobacterium, immunostimulators, zincproline, detergent, modified bacterial products, Freund's CompleteAdjuvant, Freund's Incomplete Adjuvant, Gerbu Adjuvant, Ribi's Adjuvant,and any combination thereof.
 3. The immunogenic composition of claim 1,wherein the isolated Fusobacterium necrophorum peptide is recombinantlyproduced or chemically synthesized.
 4. The immunogenic composition ofclaim 1, wherein the Fusobacterium necrophorum peptide induces an immuneresponse specific for a Fusobacterium species when administered to ananimal.
 5. The immunogenic composition of claim 1 further comprising aleukotoxin immunogen.
 6. The immunogenic composition of claim 1 furthercomprising a non-adhesin polypeptide operatively linked to the isolatedadhesin protein.
 7. The immunogenic composition of claim 6, wherein thenon-adhesin polypeptide is the glutathione S-transferase (GST) fusionprotein.
 8. An immunogenic composition comprising: a. a Fusobacteriumnecrophorum peptide having a sequence selected from the group consistingof SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5; and, b. apharmaceutically acceptable carrier comprising a component selected fromthe group consisting of adjuvants, stabilizing agents, preservatives,antibacterial agents, antifungal agents, adsorption delaying agents, andany combination thereof.
 9. The immunogenic composition of claim 8,wherein the adjuvant is selected from the group consisting ofsurfactants, oil, mycobacterium, immunostimulators, zinc proline,detergent, modified bacterial products, Freund's Complete Adjuvant,Freund's Incomplete Adjuvant, Gerbu Adjuvant, Ribi's Adjuvant, and anycombination thereof.
 10. The immunogenic composition of claim 8 furthercomprising a leukotoxin immunogen, or a non-adhesin polypeptideoperatively linked to the isolated Fusobacterium necrophorum peptide.11. A kit comprising: a. a Fusobacterium necrophorum peptide having asequence selected from the group consisting of SEQ ID No. 3, SEQ ID No.4, and SEQ ID No. 5; b. a pharmaceutically acceptable carrier comprisinga component selected from the group consisting of adjuvants, stabilizingagents, preservatives, antibacterial agents, antifungal agents,adsorption delaying agents, and any combination thereof; c. a container;and d. instructions for use of the kit components.
 12. An immunogeniccomposition comprising: a. a Fusobacterium necrophorum peptide having asequence selected from the group consisting of SEQ ID No. 3, SEQ ID No.4, and SEQ ID No. 5; and, b. a component selected from the groupconsisting of adjuvants, stabilizing agents, preservatives,antibacterial agents, antifungal agents, adsorption delaying agents, andany combination thereof.
 13. The immunogenic composition of claim 12,wherein the adjuvant is selected from the group consisting of Freund'sComplete Adjuvant and an adjuvant comprising block copolymer, squalene,and a microparticulate stabilizer.